Cereal grains, such as corn, Indian millet, barley and wheat, which are most frequently used in feed, usually provide 30-60% of the amino acid requirement. Thus, in order to satisfy the remaining requirements and maintain the balance between essential amino acids, additional provision of amino acids is required. Also, all feeds contain certain amounts of vitamins, and if any vitamin is not provided in a sufficient amount, a deficiency of the vitamin can be occurred. Thus, vitamins should additionally be provided to maintain their proper levels, like amino acids. Amino acids are the most expensive among feed components, and efficient provision of amino acids can be considered as one of factors that determines the overall ability to produce livestock. Particularly, L-lysine and L-threonine among amino acids frequently become the first amino acids that limit the growth of livestock. L-lysine and L-threonine may be produced by a fermentation process using microorganisms, and the L-lysine and L-threonine produced by the fermentation process are added to feed after purification and concentration. Microorganisms that are typically used in the fermentation of L-lysine are Corynebacterium sp. or Escherichia coli, and many examples that produced L-lysine by genetically engineering the microorganisms have been reported (Korean Patent No. 10-0930203 and No. 10-0924065, and U.S. Pat. No. 7,871,801).
Currently, efforts are being continuously made to increase the production of L-lysine or L-threonine by modified microorganisms using genetic engineering methods. However, with the growth of the industry, the ability to provide increased amounts of L-lysine or L-threonine is required, and thus efforts are being made to develop methods capable of producing L-lysine or L-threonine in a more effective and economical way.
Although vitamins are required in small amounts, they are essential organic compounds that must be provided for the maintenance of normal metabolic functions, growth, reproductive functions and health of livestock. Among vitamins, riboflavin (vitamin B2) is a water-soluble vitamin that is biosynthesized in various species of microorganisms and all kinds of plants, but it is not biosynthesized in the body of vertebrates, including humans, and thus is required to be provided by external sources. A deficiency of riboflavin can cause anestrus and reproductive failure in pigs (Biol. Reprod. (1981) 25:659-665, J. Anim. Sci. (1984) 59:1567-1572). In fowls, it can cause problems in nerves, particularly sciatic nerves and brachial nerves and can adversely affect the growth of embryos, resulting in the death of embryos (the Korean Feeding Standard for Poultry, 2002, the Korean Ministry of Agriculture and Forestry). Thus, riboflavin has been used as a feed additive for the growth of livestock, and particularly, concentrated riboflavin itself has been used as feed.
The current worldwide production of riboflavin is 6,000 tons per year, of which 75% is used as feed additives and the remainder is used as foods and pharmaceuticals. In riboflavin production, a chemical synthesis method and a microbial fermentation method are used. In a chemical synthesis method, high-purity riboflavin is produced from a precursor such as D-ribose by a multi-step process. The chemical synthesis method has a disadvantage in that the starting material is expensive and thereby increases the production cost. For this reason, a method of producing riboflavin by microbial fermentation was developed. The microbial fermentation method is a method in which either a microorganism that produces riboflavin is isolated from nature or a microorganism mutated by a genetic engineering method or a chemical/physical method so as to overproduce riboflavin is cultured under suitable conditions, and then riboflavin is isolated from the culture. In recent years, the fermentation method has been primarily researched, because it is price-competitive and environmentally friendly. Riboflavin produced by the fermentation method is added to feed after purification and concentration.
A typical method of producing riboflavin using the yeast Candida famata is disclosed in U.S. Pat. No. 5,231,007. In the industrial production of riboflavin, Eremothecium ashbyii and Ashbya gossypii (WO No. 95/26406), which are belong to Ascomycetes, are most frequently used. In addition, the bacterium Bacillus subtilis was also reported as a strain that can be used for producing riboflavin. Many examples that produced riboflavin by genetically engineering the above bacterium have been reported (EP No. 0821063, U.S. Pat. Nos. 5,837,528, and 5,334,510), and the present inventors also produced riboflavin using the above bacterium (Korean Patent No. 10-0542573). In addition, an example that produced 4.5 g/L of riboflavin using a microorganism engineered to overexpress a riboflavin biosynthesis-related enzyme gene was also reported (J. Ind. Microbiol. Biotechnol. (1999), 22:8-8).
The requirements for components in animal feed are about 1-5 g/kg of L-lysine, about 0.6-3.3 g/kg of L-threonine, and 2-4 mg/kg of riboflavin, which is about 0.1% of the requirement of L-lysine (NRC. 1998. National Academy of Sciences—National Research Council, Washington, D.C.). However, L-lysine and riboflavin are separately produced by fermentation processes, are subjected to purification and concentration processes before their addition to feed, and are individually transferred to a feed compounding plant. For this reason, they can increase the production cost of feed. If the concentration of riboflavin in a microorganism that produces both L-Lysine and riboflavin reaches about 0.1% of the concentration of L-lysine, addition of the microbial culture can satisfy the requirements for feed additives, but attempts to achieve this have not yet been reported.
In the case of Corynebacterium sp. microorganisms, riboflavin is biosynthesized through two pathways from ribulose 5-phosphate (Ru5P), which is a pentose-phosphate pathway (PPP) product, and guanosine triphosphate (GTP) that is a purine metabolism product. In the biosynthesis of riboflavin, a gene family consisting of GTP cyclohydrolase II (RibA) gene, pyrimidine deaminase-reductase (RibG) gene, riboflavin synthase subunit alpha (RibC) gene and riboflavin synthase subunit beta (RibH) gene (hereinafter referred to as “riboflavin biosynthesis gene family”) is involved. The riboflavin biosynthesis gene family forms an operon (rib operon) with ribulose-5-phosphate-3-epimerase (Rpe, NCgl1536) that is involved in the pentose-phosphate pathway, and both the Rpe and the RibA compete in the use of Ru5P as a substrate (FIG. 1). In other words, Rpe biosynthesizes D-xylose-5-phosphate from Ru5P to mediate an intermediate process in which a metabolic product produced in the pentose-phosphate pathway enters the glycolytic pathway, and RibA biosynthesizes 3,4-dihydroxy-2-butanone-4-phosphate that is an intermediate of riboflavin biosynthesis (KEGG, Kyoto Encyclopedia of Genes and Genomes, [at the world wide web address: genome.jp/kegg]).
The pentose-phosphate pathway in Corynebacterium is the major source of reducing power (NADPH) that is involved in lysine biosynthesis, and the direct correlation between the regeneration of NADPH and L-lysine biosynthesis has been reported in the literature (Wittmann and Heinzle, Microbiol 68:5843-5849, 2002; Marx et al., J Biotechnol 104:185-197, 2003; Ohnishi et al., Microbiol Lett 242:265-274, 2005).
The pentose-phosphate pathway is catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase (6PGD), Rpe, ribose-5-phosphate isomerase (RpiA), transketolase and transaldolase, and the final metabolic product produced by this pathway enters the glycolytic process.
As a result of studies on the enforcement of the pentose-phosphate pathway, EP 01941065 (June, 2001) and EP 02781875 (December, 2002) disclose an enzyme variant which directly produce NADPH, which has negative feedback resistance. In addition, inventions relating to lysine-producing Corynebacterium strains that overexpress transketolase and transaldolase were disclosed (EP 1109915, EP 1179076, and EP 1179084). Moreover, an increase in the production of L-lysine in a Corynebacterium strain that overexpresses Rpe or RpiA was reported (DE10037611 and DE10037612).
Thus, it can be seen that as the production of L-lysine in a Corynebacterium strain increases, the dependence of the strain on the pentose-phosphate pathway increases. However, the riboflavin biosynthesis pathway that is the key element of the present invention is derived from the pentose-phosphate pathway, and thus if a carbon flow to the riboflavin biosynthesis pathway is enhanced, a carbon source to be introduced into the glycolytic pathway through the pentose-phosphate pathway will leak, resulting in a decrease in the L-lysine production yield per unit of carbon source supplied. In other words, it can be considered that the riboflavin biosynthesis pathway in an L-lysine-producing strain that requires a sufficient carbon flow in the pentose-phosphate pathway is competitive with the pentose-phosphate pathway. Also, if a carbon flow that is introduced into the riboflavin biosynthesis pathway increases, the production of L-lysine can be adversely affected.
Thus, microorganisms that simultaneously produce L-lysine and riboflavin can be present in nature, but the production yields of L-lysine and riboflavin can be competitive with each other. For this reason, attempts to increase the production of riboflavin in an industrial microorganism, which produces a large amount of L-lysine, to an industrially useful level, have not yet been reported.